Antibiotic resistance poses a significant threat to public health. The emergence of several vancomycin-resistant Staphylococcus aureus strains in recent years is particularly frightening because S. aureus is a highly virulent pathogen. There is a pressing need for new strategies to treat resistant Gram positive infections. The long term goal of the proposed research is to evaluate a largely unexplored metabolic pathway as a target for antimicrobial chemotherapy. This pathway, found in many pathogenic Gram positive bacteria, involves the biosynthesis of wall teichoic acids (WTAs). Wall teichoic acids are surface-associated anionic polymers. In some organisms these polymers are essential for survival;in S. aureus, they function as virulence factors that play a critical role in the establishment and spread of infection. Therefore, strategies to disrupt wall teichoic acid biosynthesis may have therapeutic utility. The specific aims of the research include: I) Elucidating the enzymology of key WTA biosynthetic enzymes from B. subtilis, the major Gram positive model organism. Chemical methods, approaches, and tools will be developed to study TagA, TagB, and TagF, three enzymes involved in WTA biosynthesis in B. subtilis 168. Unusual features of these enzymes will be explored. II) Characterizing the pathway for WTA biosynthesis in S. aureus using a combination of genetics and in vitro biochemistry. The approaches developed to study the 6. subtilis enzymes will be applied to characterize the S. aureus enzymes. Unresolved questions about WTA biosynthesis in S. aureus will be addressed. III) Establishing a multi-target high throughput screen for wall teichoic acid biosynthesis. An in vitro screen for several successive enzymes involved in WTA biosynthesis will be established and implemented, and the hits will be evaluated against B. subtilis and S. aureus targets. The experiments outlined in this proposal will lay the groundwork for evaluating wall teichoic acid biosynthesis as a target for antimicrobial chemotherapy and will provide fundamental information about the WTA pathway in S. aureus and about some of the more interesting WTA enzymes.